reaction mass of sodium 1-amino-4-[[3,5-bis[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate and sodium 1-amino-4-[[3-[[(chloroacetyl)amino]methyl]-2,4,6-trimethylphenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

other: data on Acid Blue 225_constituent 1

Adequacy of study:

key study

Study period:

From April 06 to May 26, 2016

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Justification for type of information:

The experiment was conducted on one of the substance components (details are given in the document attached to IUCLID section 13.2).

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

March 2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

None

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the bulk test preparation on Day 0 and from the pooled replicates on Day 7 for quantitative analysis. All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Details on test solutions:

A nominal amount of test item (200 mg) was dissolved in culture medium and the volume adjusted to 2 liters to give a 100 mg/l test concentration from which a series of dilutions was made to give further test concentrations.

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Strain: Canadian Phycological Culture Centre, University of Waterloo, Ontario, Canada.
- Method of cultivation: cultures were maintained in the laboratory by the periodic replenishment of culture medium. The culture was maintained in the laboratory at a temperature of 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) for at least 7 days prior to the start of the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test temperature:

24 ± 1 °C

pH:

6.5 ± 0.2

Nominal and measured concentrations:

Nominal 6.25, 12.5, 25, 50 and 100 mg/l

Details on test conditions:

TEST SYSTEM
- No. of colonies per vessel: 3 colonies
- No. of fronds per colony: total 12 fronds.
- No. of vessels per concentration: 3 replicates.
- No. of vessels per control: 3 replicates.

TEST MEDIUM / WATER PARAMETERS
- Preparation of dilution water: culture medium will be prepared in reverse osmosis purified water (Elga Optima 15+ or Elga Purlab Option R-15 BP).
- pH: the pH was adjusted, if necessary, to 6.5 ± 0.2 with either 1M HCl or NaOH.
- Composition: NaNO3 85 mg/l, KH2PO4 13.4 mg/l, MgSO4.7H2O 75 mg/l, CaCl2.2H2O 36 mg/l, Na2CO3 20 mg/l, H3BO3 1.0 mg/l, MnCl2.4H2O 0.20 mg/l, Na2MoO4.2H2O 0.010 mg/l, ZnSO4.7H2O 0.050 mg/l, CuSO4.5H2O 0.0050 mg/l, Co(NO3)2.6H2O 0.010 mg/l, FeCl3.6H2O 0.84 mg/l, Na2-EDTA.2H2O 1.40 mg/.

OTHER TEST CONDITIONS
- Photoperiod: nstant illumination.
- Light intensity and quality: intensity approximately 7000 lux.

EFFECT PARAMETERS MEASURED
The number of fronds present in each test and control culture was recorded on days 0, 2, 5 and 7 along with observations on frond size, appearance, root length and number of colonies present.
In addition the dry weight of the fronds in each control and treatment group was determined on day 7. At the start of the test six replicate samples of fronds identical to those used to inoculate the test vessels were taken and the dry weight determined. At the end of the test the dry weight of colonies from each control and test vessel was determined by blotting the colonies dry and drying at 60 °C to constant weight.

RANGE-FINDING STUDY
- Test concentrations: 1.0, 10 and 100 mg/l
- Exposure period: 7 days.
- Replicates: 2 replicate flasks were prepared for each control and test concentration.
- Incubation: flasks were then incubated at 24 ± 1 ºC under continuous illumination (intensity approximately 7000 lux) for 7 days.
- Test item stability: a sample of each test concentration was taken for chemical analysis on Day 2 (fresh media) and Day 5 (old media). An additional sample of each test concentration was prepared on Day 0 and incubated alongside the test until Day 7 in order to determine stability over the entire test duration.

VALIDITY OF THE TEST
The results of the test are considered valid if the following performance criterion is met: the doubling time of frond numbers in the controls must be less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0.275 d-1.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

ErC10 (7d): 36 mg/l, based on frond number
ErC10 (7d): 30 mg/l, based on dry weight
EyC10 (7d): 8.7 mg/l, based on frond number
EyC10 (7d): 7.9 mg/l, based on dry weight

MEASURED CONCENTRATIONS
Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to be near nominal with the exception of the 6.25 mg/l test sample on Day 7 where a measured concentration of 66 % of nominal was obtained. Given that, the concentration was below the No Observed Effect Concentration (NOEC) this was considered to have had no adverse effect on the outcome of the test and as such it was considered appropriate to calculate the results based on nominal test concentrations only.

RANGE FINDING TEST
The frond counts and percentage inhibition of growth values from the exposure of Lemna minor to the test item during the range-finding test showed no significant effect on growth at the test concentrations of 1.0 and 10 mg/l. However, growth was observed to be reduced at 100 mg/l.
Chemical analysis of the 10 and 100 mg/l test preparations on Days 2, 5 and 7 showed near nominal concentrations were obtained indicating that the test item was stable under test conditions.

Inhibition of Average Specific Growth Rate and Yield Based on Dry Weight

Nominal Concentration (mg/l)		Average Specific Growth Rate		Yield
		(0 – 7 Day)	% Inhibition	(0 – 7 Day)	% Inhibition
Contol	Mean	0.423	-	16.8	-
	SD	0.034		4.4
Test item 6.25	Mean	0.433	[2]	17.9	[7]
	SD	0.023		3
Test item 12.5	Mean	0.395	7	13.4	20
	SD	0.018		1.7
Test item 25	Mean	0.395	7	13.4	20
	SD	0.014		1.4
Test item 50	Mean	0.362	14	10.4	38
	SD	0.004		0.4
Test item 100	Mean	0.38	10	12.2	27
	SD	0.034		3.2

SD – Standard Deviation

[Increase in growth as compared to the controls]

Frond Numbers and Percentage Inhibition of Growth from the Range-finding Test

Nominal Concentration (mg/l)	Number of Fronds				Average Specific Growth Rate		Yield
	Day 0	Day 2	Day 5	Day 7	(0 – 7 Day)	% Inhibition	(0 – 7 Day)	% Inhibition
Control R1	9	21	59	116	0.365	-	107	-
Control R2	9	21	64	132	0.384		123
Mean	9	21	62	124	0.375		115
Test item 1 mg/l R1	9	22	72	154	0.406	[8]	145	[25]
Test item 1 mg/l R2	9	21	67	151	0.403		142
Mean	9	22	70	153	0.405		144
Test item 10 mg/l R1	9	21	65	126	0.377	2	117	6
Test item 10 mg/l R2	9	21	50	107	0.354		98
Mean	9	21	58	117	0.366		108
Test item 100 mg/l R1	9	22	56	84	0.319	15	75	35
Test item 100 mg/l R2	9	19	46	83	0.317		74
Mean	9	21	51	84	0.318		75

R1 – R2 = Replicates 1 and 2

[Increase in growth compared to controls]

Validity criteria fulfilled:

yes

Remarks:

the doubling time of the control cultures was 1.61 days in line with the OECD Guideline that states the doubling time should be less than 2.5 days

Conclusions:

ErC50 (7d) > 100 mg/l (nominal), both based on frond number and based on dry weight
EyC50 (7d) > 100 mg/l (nominal), both based on frond number and based on dry weight

Executive summary:

A study was performed to assess the effect of the test item on the growth of the freshwater plant Lemna minor. The method followed that described in the OECD Guideline No. 221 “Lemna sp. Growth Inhibition Test (March 2006)”. Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at concentrations of 6.25, 12.5, 25, 50 and 100 mg/l (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of 24 ± 1 °C. The number of fronds in each control and treatment group was recorded on days 0, 2, 5 and 7 along with observations on plant development.

Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to be near nominal with the exception of the 6.25 mg/l test sample on Day 7 where a measured concentration of 66 % of nominal was obtained. Given that the concentration was below the No Observed Effect Concentration (NOEC) was considered to have had no adverse effect on the outcome of the test and as such it was considered appropriate to calculate the results based on nominal test concentrations only.

The EC50 values for both growth rate and yield resulted to be higher than 100 mg/l (nominal), in both the cases of effects based on frond number and dry weight.

Conclusion

ErC50 (7d) > 100 mg/l (nominal), both based on frond number and based on dry weight

EyC50 (7d) > 100 mg/l (nominal), both based on frond number and based on dry weight

Description of key information

Not harmful/toxic to aquatic plants other than algae (Er/yC50 (7d) > 100 mg/l (nominal), both based on frond number and based on dry weight)

Additional information

There is no data on the toxicity potential of Acid Blue 225 to aquatic plants, therefore the available data on Acid Blue 225_constituent 1 has been taken into consideration. Acid Blue 225_constituent 1 is one of the Acid Blue 225 constituents. The data can be considered as adequate and the approach can be considered as suitable (details are given in the document attached to IUCLID section 13.2).

A study was performed to assess the effect of the Acid Blue 225_constituent 1 on the growth of the freshwater plant Lemna minor. The method followed that described in the OECD Guideline No. 221 “Lemna sp. Growth Inhibition Test (March 2006)”. Following a preliminary range-finding test, Lemna minor was exposed to an aqueous solution of the test item at concentrations of 6.25, 12.5, 25, 50 and 100 mg/l (three replicate flasks per concentration) for a period of 7 days, under constant illumination at a temperature of 24 ± 1 °C. The number of fronds in each control and treatment group was recorded on days 0, 2, 5 and 7 along with observations on plant development. Chemical analysis of the test preparations on Day 0 (fresh media) and Day 7 (old media) showed measured test concentrations to be near nominal with the exception of the 6.25 mg/l test sample on Day 7 where a measured concentration of 66 % of nominal was obtained. Given that the concentration was below the No Observed Effect Concentration (NOEC) was considered to have had no adverse effect on the outcome of the test and as such it was considered appropriate to calculate the results based on nominal test concentrations only. The EC50 values for both growth rate and yield resulted to be higher than 100 mg/l (nominal), in both the cases of effects based on frond number and dry weight.